Prevalence and epidemiological characteristics of methicillin-resistant Staphylococcus aureus isolated from cattle in Bangalore India as a part of the One Health approach

In India, limited studies are available on the epidemiological aspects of methicillin-resistant Staphylococcus aureus (MRSA) infections in both animal and human settings. Herein, we investigated the prevalence, antimicrobial resistance profile and molecular characteristics of MRSA isolates recovered from cattle using the One Health approach. Out of 66 mecA-positive staphylococci, species-specific multiplex PCR detected 24 % (n=16) of isolates as MRSA. Maximum antibiotic resistance was seen against cloxacillin (94 %, n=15) and least for enrofloxacin and cephalothin (each 13 %, n=2). Overall, 13 % (n=2) of MRSA isolates were multidrug-resistant. Molecular characterization by SCCmec typing identified 88 % (n=14) of MRSA isolates as type V. Twelve isolates (75 %) belonged to novel spa-type t17242, of which 67 % (n=8) belonged to agr type I. MLST analysis revealed ST 1687 (50 %, n=8) as the most predominant sequence type. Circulation of different MRSA clones among the cattle populace offers a risk of transmission to humans through direct contact, food chain or environmental contamination. Thus, continuous monitoring of MRSA strains is imperative for early diagnosis and for establishing effective treatment strategies to restrain the disease burden caused by MRSA infections.


INTRODUCTION
Methicillin-resistant Staphylococcus aureus (MRSA) is considered a significant threat to both human and animal health [1].Recently, there have been growing reports of MRSA causing clinical illnesses in various animals, such as companion animals, pigs and dairy cattle [2].The mechanism behind methicillin resistance in MRSA involves the mecA gene, which encodes a modified penicillin-binding protein (PBP2a) with low affinity for β-lactam antibiotics [3].As a result, PCR-based detection of the mecA gene is considered the 'gold standard' for detecting methicillin resistance in staphylococcus [4].
Besides the mecA gene, there are other determinants of methicillin resistance, such as mecC (formerly known as LGA251), which shares 69 % nucleotide identity with mecA.It is believed that mecC MRSA emerged in animals, particularly ruminants, and subsequently spread to humans, posing a zoonotic risk [5].Another methicillin-resistance determinant, mecB, has recently been discovered in Macrococcus caseolyticus.Given that macrococcal and staphylococcal species share the same hosts, it is likely that mobile genetic elements could facilitate the transfer of the mecB gene between these closely related genera [6].Notably, methicillin-resistant M. caseolyticus strains from bovine and canine sources have also been found to carry a novel mecD gene, which confers resistance to all classes of β-lactams, including anti-MRSA cephalosporin [7].
Given the potential for MRSA to be transmitted from animals to humans, it is necessary to conduct epidemiological surveillance to track the emergence and incidence of MRSA as well as epidemic S. aureus clones, which may pose zoonotic risks [8].Studies have demonstrated that methicillin-susceptible S. aureus (MSSA) can acquire methicillin-resistance determinants via the staphylococcal cassette chromosome mec (SCCmec) element, resulting in MRSA [9].To better understand the epidemiological characteristics of MRSA strains, it is important to use appropriate molecular methods capable of monitoring changes over time [10][11][12].Herein, we investigated the prevalence, antimicrobial resistance profile and molecular epidemiological characteristics of MRSA isolates recovered from cattle in Bangalore India using the One Health approach.

Sample collection and bacterial isolation
A total of 666 samples were collected from various organized (involves planned activities) and unorganized (no planned activities) cattle farms located in and around Bangalore between 2015 to 2017.The samples comprised cattle milk (n=371), cattle nasal swabs (n=109), extramammary site (n=90), cattle wound (n=30), animal handlers hand swabs (n=32) and environmental swabs (n=34).The environmental samples included feed trough (n=13), floor of cattle shed (n=15), milking machine (n=4) and supplied water (n=2).The disparity in the sample size among different sources was primarily due to resource constraints and logistical challenges.
Aseptic procedures were followed during the collection of milk samples, which involved cleaning the udder and teats with 70 % ethyl alcohol.The first few streams of milk were discarded, and 5 ml of milk was then collected in a sterile collection bottle containing 2 ml of Brain Heart Infusion (BHI) broth (Himedia Laboratories, Mumbai, India).All other samples (nasal, extramammary site, wound, animal handlers hand, environmental samples) were collected using sterile cotton swabs dipped in sterile tubes containing BHI broth.The samples were transported to the bacteriology laboratory of ICAR-NIVEDI, Bangalore, India, within 2-4 h.On arrival, the samples were immediately enriched in Mannitol salt broth (MSB) and incubated at 35-37 °C for 18-24 h.Each sample was then cultured on staphylococcus agar 110 (S110) (HiMedia, Mumbai, India) and incubated for 24 h at 37 °C.The colonies appeared as small, tiny, creamy, circular forms and some pathogenic forms also formed pigmented colonies from yellow to orange.Based on the colony morphology and pigmentation, a total of 2-3 colonies from each S110 agar plate were subsequently processed in BHI agar to yield pure cultures of isolates.The pure cultures were stored in 15 % glycerol for further downstream processing.Staphylococcus was tentatively identified based on colony morphology, pigment production, Gram staining, catalase and oxidase tests as per the standard protocol [13].

Molecular identification of Staphylococcus spp
The DNA extracted from the culture isolates was subjected to multiplex PCR for the simultaneous detection of genus staphylococcus and methicillin resistance determinants (i.e.mecA and mecC genes) as described previously [14][15][16].The mecA and/or mecC positive staphylococcus isolates were subjected to species-specific multiplex PCR targeting five important staphylococcus species as detailed previously [17,18].ATCC 33591 (mecA positive S. aureus) and BH32 (mecC positive S. saprophyticus) were used as positive control strains.BH32 strain is from the Shome Laboratory bacterial strain inventory (GenBank accession no.MG334392).

Antibiotic susceptibility testing
Kirby-Bauer's disc diffusion method was used to test the mecA-positive S. aureus isolates against 12 antibiotics representing five distinct antimicrobial classes (Table 1) [19].The rationale for the choice of antibiotics examined was based on their widespread use in food animals.The isolates were classified as either susceptible, intermediate or resistant according to the guidelines of the Clinical and Laboratory Standards Institute [20].S. aureus ATCC 25923 served as the quality control strain, while isolates that were non-susceptible to at least one agent in three or more antimicrobial classes were defined as multidrug-resistant (MDR) [21].Additionally, the minimum inhibitory concentration (MIC) of cefoxitin was determined using the E-test (Himedia Laboratories).Isolates with an MIC value of ≥8 µg ml −1 for cefoxitin were considered methicillin-resistant [22] (Table 1).S. aureus ATCC 29213 was used as a quality-control strain for the MIC method.

Molecular epidemiological typing of methicillin-resistant Staphylococcus aureus isolates
The MRSA isolates were subjected to PCR-directed SCCmec typing as previously described by Kondo et al. [23] using two multiplex PCRs -one for ccr type assignment and another for mec class assignment [23].Further, all the MRSA isolates were characterized by spa typing [24].Amplification of the spa repeat region was performed using a set of designed primers 1067F (5′-ACGTAACGGCTTCATCCA-3′) and 1704R (5′-TCCA CCAA ATAC AGTT GTACCG-3′).The cycling conditions involved initial denaturation at 94 °C for 5 min, 30 cycles of 94 °C for 30 s, 56 °C for 30 s, and 72 °C for 45 s followed by a final extension at 72 °C for 5 min.The PCR assay was performed in a 25 µl reaction volume containing 1×PCR buffer, 1.5U DNA Taq polymerase, 2 mM MgCl 2 , 200 µM deoxynucleotide triphosphates; (Fermentas, Glen Burnie, MD, USA), 0.5 µM of each primer and 50 ng template DNA.Amplified products were sequenced (Eurofins, Bangalore, India), and the resulting spa type and spa repeats were assigned by submitting the data to the S. aureus spa type database (http://spaserver.ridom.de/).

RESULTS AND DISCUSSION
Periodic surveillance to monitor antimicrobial resistance patterns, specifically methicillin resistance in staphylococci obtained from dairy cattle could be a crucial strategy in ascertaining the emergence and spread of drug-resistant S. aureus.Studies have highlighted various drivers of MRSA spread within cattle-rearing settings [26].These drivers can include factors such as intensive farming practices, inadequate biosecurity measures, overcrowding, inappropriate antibiotic use, and the proximity of livestock to humans.These factors create an environment that promotes the transmission and persistence of MRSA strains among cattle populations.Further, considering the broader One Health context, MRSA strains originating from livestock can pose a significant risk to human health [27].Transmission of MRSA from animals to humans can occur through direct contact, occupational exposure, consumption of contaminated food products, or environmental contamination.By investigating the drivers of MRSA spread within cattle-rearing settings and their implications for human health, we can better assess the overall risk and develop appropriate cost-effective control measures.Herein, we set out to study the frequency and epidemiological features of MRSA isolates recovered from cattle in a One Health framework.
As shown in Fig. 1, multiplex PCR identified a total of 66 isolates as mecA-positive staphylococci.None of the isolates was mecCpositive.Using species-specific multiplex PCR, 24 % (n=16) of the mecA-positive isolates were detected as S. aureus (MRSA) (95 % CI, 0.17 to 0.30), and 76 % (n=50) as coagulase-negative staphylococcus (MRCoNS) (95 % CI, 0.69 to 0.82).Out of the 16 MRSA isolates, 15 were recovered from cattle milk samples (95 % CI, 0.81 to 1.05), and one isolate was obtained from a cattle wound sample (95 % CI, −0.05 to 0.18).Conversely, no MRSA isolate was recovered from the other samples we collected.In  European countries, the prevalence rate of bovine MRSA varies between 0.4-17 % [28,29].Among Asian countries, India and China report the prevalence rate of bovine MRSA to be 13 and 48 %, respectively [30,31].These differences in the frequencies of resistance could be attributed to variations in the animal populations examined, dissimilarity in farm management systems, different methodologies and sampling strategies implemented, variety of drug usage, and the geographical location of the studies.
While cefoxitin is a possible alternative for identifying methicillin resistance in a resource-limited situation, the usefulness of this approach is restricted by the heterogeneity in the expression of methicillin resistance in S. aureus.Ideally, all mecApositive S. aureus isolates should be resistant to cefoxitin, but this is not always the case.We identified four mecA-positive isolates, which were sensitive to cefoxitin either by disc diffusion or MIC method.The heterogeneity may complicate the detection of MRSA, as phenotypic detection is based on various variables like temperature, pH, the osmolarity of the medium, etc. Hence detecting the mecA gene by PCR remains the gold standard test for the detection of MRSA.Similar observations have been reported by other groups [32][33][34].
Furthermore, among all the antibiotics tested, enrofloxacin was the only antibiotic to which most of the isolates (88 %) were susceptible (95 % CI, 0.72 to 0.87).In contrast, a high level of resistance was observed against cloxacillin (94 %), oxacillin (88 %), ampicillin (81 %) and penicillin G (81 %), which is in accordance with previous reports [30,35].The resistance pattern of MRSA isolates against other antibiotics is depicted in Table 1.The emergence of MDR MRSA is becoming a significant public health concern [36].Overall, multidrug resistance was observed in 13 % (n=2) of the total MRSA isolates (95 % CI, −0.03 to 0.28) (Table 1).MIC testing of MRSA isolates against cefoxitin revealed six resistant isolates with MIC values ranging between eight to ≥256 µg ml −1 .The rise of such strains might be attributed to the indiscriminate use of antibacterial drugs without prior drug susceptibility testing or selection pressure of antimicrobials on pathogens [37].Consequently, the need for more effective measures against the unregulated use of antibiotics is necessary to prevent the development of MDR strains.
The majority of our MRSA isolates belonged to SCCmec type V (88%, n=14) (95 % CI, 0.71 to 1.03) (Table 2).Independent studies from Italy and Germany reported >75 % of the MRSA isolates recovered from dairy herds harboured SCCmec type V [38,39].Studies from Europe warranted that MRSA isolates with SCCmec type IV or V play a vital role in clinical or subclinical bovine mastitis [40].The significance of the SCCmec type V in bovine MRSA has been interrelated with the resistance to zinc [41].It is described that metal resistance genes that are frequently associated with the animal origin bacteria can promote the bacteria to fix antimicrobial resistance genes, which can be transmitted to humans through contaminated food or environment [42].On the basis of spa typing, the MRSA isolates were distributed into three known spa types (t045, t267, and t359) and two novel spa types (t17242 and t18033) (Table 2).The majority (75 %, n=12) of the MRSA isolates belonged to t17242, with 83 % (n=10) of them belonging to SCCmec type V. Two t17242 isolates were non-typeable for SCCmec, which may be due to genetic variation in the target genes.Apparently, there is a high level of unpredictability in creating new SCCmec types in staphylococci [43], which we could not target with our assay.We found spa type t045 in only one MRSA isolate; whereas, a study from the US [44], found t045 in 73 % of isolates.The prevalence of spa types among S. aureus isolates varies in different areas around the world.These data are useful for defining the geographical spread of the predominant spa types of S. aureus, interpreting relative frequencies, and understanding molecular epidemiological dynamics of S. aureus transmission.
Moreover, MLST and agr typing represent the key markers to study the epidemiology of S. aureus.MLST analysis revealed ST 1687 (50 %, n=8) as the most common ST among MRSA isolates (95 % CI, 0.37 to 0.62), with seven of them belonging to spa type t17242 and SCCmec type V.In addition, five novel STs were detected viz., ST 5217 (n=2) and one each of ST 5216, ST 5218, ST 5219 and ST 5220 (Table 2).Interestingly, bovine MRSA isolates belonging to a novel ST 1687 were first reported from India [45].Phylogenetic analysis of MRSA isolates (Fig. 2) revealed that ST 2668 exhibited the maximum divergence in the population framework.The findings highlight the potential significance of ST 2668 as a major source of spread within the study site.
S. aureus derived from hospitals and cattle was shown to have a high prevalence of agr type I [46,47].We detected agr type I in 63 % (n=10) of MRSA isolates (95 % CI, 0.45 to 0.79), which is in accordance with earlier reports [48,49], indicating agr type I as the frequent group among the S. aureus isolates.Moreover, 67 % (n=8) of spa type t17242 belonged to agr type I.Ten isolates with agr type I were distributed into six STs and three spa types (ST 5218-t17242, ST 1687-t17242, ST 5220-t17242, ST 2668-t17242, ST 5216-t359 and ST 5217-t267), with the most predominant clone being ST 1687-t17242-agr type I. Studies from South Korea by Lim et al. detected ST 72-t324-SCCmec type IV as the most predominant clone in cow milk, farmers and farm environment [50].In India, the predominant clone of MRSA causing bovine mastitis in regions of Telangana, Tamil Nadu and Andhra Pradesh was found to be ST 72-t17287, with the majority of the isolates carrying SCCmec type III [45].Previous studies have shown that S. aureus strains belonging to agr group I can invade epithelial cells of the mammary gland, demonstrating that agr type I strains can be the major etiological agent in mastitis [51].the development of targeted interventions to curb its spread effectively.We have now incorporated these findings in our revised manuscript to further enrich the discussion and broaden the impact of our study.
-Line 32-33, Line 84-85-The isolates are from cattle and other sources within the production setting.One Health should reflect in objective.
Response:The study employed a One-Health approach, aiming to collect comprehensive information on MRSA by analyzing various sample types, including animal, human, and environmental samples.However, despite our efforts, we were only able to recover MRSA isolates from cattle samples, specifically from cattle milk and cattle wound.Consequently, this rationale supports the statement that our investigation focused on examining the prevalence, antimicrobial resistance profile, and molecular epidemiological characteristics of MRSA isolates isolated from cattle.However, we have now modified the statements as suggested.
Lines Comments: The epidemiological study by Venugopal et al describes the prevalence of antibiotic resistance in Staphylococcus aureus strains isolated from cattle in India.The study is well written and has been performed using a rigorous methodology that is classical for such a study regarding S. aureus.The authors have presented the results in a clear manner with a robust comparison of their findings in the context of other studies.The manuscript is easy to read and has clear conclusions.I have two main comments for consideration: 1.The samples numbers and their corresponding isolate ID's appear to differ between Tables 1 and 2. It would be useful to standardize the sample numbers between the tables to decrease confusion for the reader unless there is a clear reason why isolates are given different sample numbers between the two tables.2. Line 203 in the text appears to be missing some wording.

Please rate the manuscript for methodological rigour Very good
Please rate the quality of the presentation and structure of the manuscript Very good

Fig. 1 .
Fig. 1.Study flow chart illustrating the events involved in the isolation and characterization of MRSA isolates.Abbreviations: MRSA, methicillin-resistant Staphylococcus aureus; MRCoNS, methicillin-resistant coagulase-negative staphylococcus; MIC, minimum inhibitory concentration.The multiplex PCR targets the genus Staphylococcus and methicillin resistance determinants (i.e.mecA and mecC genes).

Table 1 .
Antibiotic susceptibility profile of MRSA isolates

Table 2 .
Epidemiological typing of MRSA isolates Response:Changes are done as suggested.As per the recommended nomenclature, all the gene names and species names have been italicized.The genus name (e.g., staphylococcus) is not supposed to be italicized.Your manuscript is well written but requires some minor edits and additional discussion points to improve the manuscript.Please can the authors address the reviewers comments below, adding discussion and clarification to the text where required.Additionally, please can you merge the methods sections given in the main manuscript and supplementary so that all of the methodology details are in the main body of the text.thecattlerearingsettings.For instance, all isolates presented in results were recovered from only milk and wound samples, does this mean that MRSA was not recovered from other samples?This must be clarified as it will inform dynamics of spread.Confidence interval should be included.2.There should be discussions on the drivers of MRSA spread within cattle rearing settings and how these can influence spread from a broader One Health context.Finally, the observed dynamics can inform recommended cost effective preventative solutions in cattle production areas.3.The authors mention a 'One Health' approach.The sampling strategy should reflect this approach in the main manuscript and not supplementary material.4.The authors should include MLST data analysis to provide information on the diversity and population structure of MRSA.Data on phylogenetics which is available from MLST is important for source tracking purposes.There is more value in understanding the spread of resistant isolates within the respective study sites as this information can be used to develop potential interventions.Minor comments.-Line32-33,Line84-85-Theisolatesarefromcattle and other sources within the production setting.One Health should reflect in objective.-Consistencye.g., five or 5 (Line 108, 186) -Italic scientific names e.g., Line 94, 99-103Please rate the manuscript for methodological rigour GoodPlease rate the quality of the presentation and structure of the manuscript GoodTo what extent are the conclusions supported by the data?Partially supportDo you have any concerns of possible image manipulation, plagiarism or any other unethical practices?NoIs there a potential financial or other conflict of interest between yourself and the author(s)?NoIf this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?YesReviewer 1 recommendation and comments https://doi.org/10.1099/acmi.0.000627.v1.4 © 2023 Pickering A. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License.
Comments:The study is on the prevalence, antimicrobial resistance profile, and molecular characteristics of MRSA from cattle rearing settings.I have the following comments: Major comments 1.The prevalence is not well described: The sample size for the milking machine and supplied water samples is much lower than other samples, therefore authors should explain their sample size selection.'Prevalence' appears in the title; therefore, it is important to show positive outcomes of MRSA from the different sources within